Multiple sclerosis (MS) is a demyelinating disease affecting the central nervous system, in which episodes of inflammation result in a highly variable course and progression of symptoms (Compston, A. & Coles, A., Lancet 372, 1502-1517, 2008). Onset is typically between the ages of 30-50, with higher prevalence in women and great geographic variability (Rosati, G., Neurol Sci 22, 117-139, 2001). There are a broad range of symptoms, reflecting the diverse anatomical targets of demyelination, but typical syndromes include: weakness, fatigue, loss of vision, cognitive impairment and impaired balance and coordination (Compston, supra). MS episodes are erratic in timing as well, leading to the general principle that MS lesions are disseminated in both space (location) and time (Adams, R et al., Principles of Neurology. sixth edn, (McGraw-Hill, 1997)). These symptoms can often be sufficient to make the diagnosis of MS, but magnetic resonance imaging (MRI) is also helpful, along with analysis of cerebrospinal fluid and nerve evoked potential measurements. MS typically begins with reversible neurological deficits (relapsing-remitting phase, or RRMS), that progress eventually to fixed disability in later life (secondary progressive phase)(Adams et al., supra). Like other aspects of the disease, the pattern, severity and timing of this progression can be very different among patients, with some experiencing profound disability with rapid progression at the outset (primary progressive MS or PPMS), while a small number of other patients have isolated, relatively mild symptoms.
The cause of MS is not known with certainty, but both genetic and environmental factors can affect susceptibility (Giovannoni, G. & Ebers, G., Curr Opin Neurol 20, 261-268, 2007). Prominent candidate genes include HLA DRB1 and DQB, which encode part of the human major histocompatibility complex (MHC), apolipoprotein E (APOE) and interferon gamma (IFN-g)(Giovannoni et al., supra; Kantarci, O. & Wingerchuk, D., Curr Opin Neurol 19, 248-254, 2006) The most commonly implicated environmental factors are vitamin D/sunlight, and infectious agents (including the Epstein-Barr virus, human endogenous retroviruses or HERV, and human herpesvirus-6 or HHV-6)(Giovannoni et al., supra). Although inflammation has been associated with MS, it is unclear whether demyelination is caused by a primary immune process or a neurodegenerative mechanism (Trapp, B. D. & Nave, K. A., Annu Rev Neurosci 31, 247-269, 2008). Autoimmune mechanisms in MS have been studied in depth, with CD4+ type 1 helper T-cells having been considered the main effector of the demyelination. Recently, other immune system components have also been implicated, suggesting that a more broad range of leukocytes may be involved, targeting both the myelin sheath and the axons themselves (Hemmer, B., et al., Nat Rev Neurosci 3, 291-301, 2002; Smith, T., et al., Nat Med 6, 62-66, 2000).
There is currently no cure for MS, and despite some recent progress in novel treatments, the disease remains a significant therapeutic challenge (Kieseier, B. C., et al., Curr Opin Neurol 20, 286-293, 2007). Standard treatment includes corticosteroids aimed at suppressing the inflammatory response during acute relapse, sometimes with plasmaphoresis to remove circulating antibodies from the bloodstream (Giovannoni et al., supra). Glatiramer acetate and interferon-β-1a are also used in RRMS, but are not particularly effective in PPMS, nor in altering the eventual course of MS, even with early intervention (Compston et al., supra; Kieseier et al., supra). A more specific immunotherapy uses monoclonal antibodies to target particular surface molecules involved in MS. Natalizumab binds to α4 integrin on white blood cells, thereby reducing their numbers, but due to adverse reactions of progressive multifocal leukoencephalopathy (PML), this drug is used only when other treatments have failed (Kieseier et al., supra). Other monoclonal antibodies, rituximab (an anti-CD20 antibody) and daclizumab (targeting CD-25), have a similar rationale, but again are not curative, and have other immunological side-effects.
The most common rodent model of MS consists of injecting myelin oligdendrocyte glycoprotein (MOG) to generate an autoimmune encephalitis (EAE). The EAE model exhibits an increase in mGluR5 expression in combination with decreased mGluR1a receptors in cerebellar Purkinje neurons. Treatment of EAE mice with an mGluR5 enhancer (RO0711401) improves motor coordination, but mGluR5 antagonists do not (Fazio, F. et al. Neuropharmacology 55, 491-499, 2008). EAE mice also show a transient reduction of Ca++-dependent glutamate release from cerebral cortex synaptosomes, soon after the onset of the clinical signs (Vilcaes, A. A., et al., J Neurochem 108, 881-890, 2009). At the same time, the EAE elevates mRNA levels of the glutamate transporters GLT-1 and GLAST in forebrain and cerebellum, in conjunction with the elevation in extracellular glutamate, although protein levels of these transporters are not consistent with the mRNA findings (Mitosek-Szewczyk, K., et al., Neuroscience 155, 45-52, 2008). Other animal models of MS, such as the CNTF −/− mouse, have decreased glutamate decarboxylase, among several proteins in the altered in EAE (Linker, R. A. et al., PLoS One 4, e7624, 2009). The neurological effects of EAE can also be attenuated by the gap junction blocker carbenoxolone or the glutaminase inhibitor DON (6-diazo-5-oxo-L-norleucine), likely through decreased glutamate release from microglia (Shijie, J. et al. Tohoku J Exp Med 217, 87-92 (2009). In vitro activation of microglial mGluR2 exacerbates myelin-induced toxicity, while mGluR3 and group III mGluRs activation is protective (Pinteaux-Jones, F. et al., J Neurochem 106, 442-454, 2008).
The role of glutamate in excitotoxicity is well established, but there is evidence that this neurotransmitter system can also modulate immune system function, and may therefore be a novel therapeutic target for inflammatory disorders of the nervous system (Hansen, A. M. & Caspi, R. R. Nat Med 16, 856-858, 2010). Glutamatergic excitotoxicity may also be involved in the mechanisms of neuronal damage by inflammation, since activated immune cells release glutamate (Pitt, D., et al., Nat Med 6, 67-70, 2000; Groom, A. J., et al., Ann N Y Acad Sci 993, 229-275; discussion 287-228, 2003). Decreased expression of glutamate transporters EAAT (excitatory amino acid transporter) 1 and 2 was reported in post-mortem cerebral cortex samples from patients with MS. These changes correlated with the presence of microglial infiltration and demyelination (Vercellino, M. et al., J Neuropathol Exp Neurol 66, 732-739, 2007). Glutamate transporters EAAT-1 and -2 are expressed by oligodendrocytes, the myelin-producing cells, and in MS lesions, the expression of these transporters is lost (Pitt, D., et al., Neurology 61, 1113-1120, 2003). Both glutamate and AMPA can enhance T-lymphocyte proliferation in response to MOG and MBP (myelin basic protein) exposure, and mGluR3 mRNA and protein are elevated on T-lymphocytes from patients with active MS compared to controls (Sarchielli, P. et al., J Neuroimmunol 188, 146-158, 2007). Antibodies against NMDA receptors have been detected in a case of recurrent optic neuritis with transient cerebral lesions, in both serum and cerebrospinal fluid, further supporting the hypothesis that autoantibodies against glutamate receptors in the CNS may play a role in demyelinating diseases (Ishikawa, N., et al., Neuropediatrics 38, 257-260, 2007).
Excessive glutamate, acting mainly through NMDARs and AMPARs, facilitates Ca2+ influx, which can result in excitotoxicity under pathological conditions including ischemia, trauma, hypoglycemia and epileptic seizure (Simon, R. P., et al., Science 226, 850-852, 1984; Choi, D. W. Trends Neurosci. 18, 58-60, 1995). The inhibition of Ca(2+)-permeable AMPA receptors may be of benefit in MS, based on the observation that mice with Gria3 mutations that do not express functional GluR3 AMPA receptor subunits, are resistant to demyelination and neurological sequelae in the EAE model (Bannerman, P. et al., J Neurochem 102, 1064-1070, 2007). In contrast, mGluR4 knockout mice are more vulnerable to the EAE model, with more T-helper cells becoming the TH17 type that produce interleukin-17 (Fallarino, F. et al., Nat Med 16, 897-902, 2010). Treatment with a selective mGluR4 enhancer appears to be protective against EAE through enhancement of regulatory Treg cells (Fallarino et al., supra). The AMPAR subunit GluR1 forms a complex with the interferon-gamma (IFN-γ) receptor that, upon activation by IFN-γ, induces cytotoxicity (Mizuno, T. et al., FASEB J 22, 1797-1806, 2008). Direct application of AMPA/kainite antagonists NBQX or MPQX reduces the acute and chronic neurological effects of EAE in rats (Smith et al., supra). The protective effects of these drugs may not involve immunological mechanisms, since NBQX did not reduce the amount of inflammation or the in vitro proliferation of activated T-cells (Pitt et al., supra). AMPA-mediated excitotoxity has also been implicated in other neurodegerative disorders, such as ALS (amyotrophic lateral sclerosis), in which motor neurons are primarily affected. Editing of the GluR2 mRNA is altered in spinal motor neurons from patients with ALS, leading to a higher proportion of Q/R site-unedited GluR-containing Ca++ permeable AMPA receptors Kwak, S., et al., Neuropathology 30, 182-188, 2010).
Functional changes in AMPARs are most often attributed to phosphorylation events mediated by cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC) and CaM kinase II (calcium-calmodulin kinase II)(Greengard, P., et al., Science 253, 1135-8, 1991; Wang, L. Y., et al., J Physiol 475, 431-7, 1994; Yakel, J. L., et al., Proc Natl Acad Sci USA 92, 1376-80, 1995; Soderling, T. R., Biochim Biophys Acta 1297, 131-8, 1996; Barria, A., et al., J Biol Chem 272, 32727-30, 1997). Recently, a variety of intracellular proteins have been reported to bind directly to AMPARs (Xia, J., et al., Neuron 22, 179-87, 1999; Dong, H., et al., Nature 386, 279-84, 1997; Osten, P., et al., Neuron 21, 99-110, 1998; Daw, M. I., et al., Neuron 28, 873-86, 2000; Allison, D. W., et al., J Neurosci 18, 2423-36, 1998). These proteins play important roles not only in receptor targeting or clustering, but also in the modulation of receptor activity and activation of signaling pathways. Protein-protein interactions have been shown to affect AMPAR trafficking and function. The best characterized AMPAR interacting proteins are those that interact with the intracellular carboxyl terminus (CT) of AMPAR subunits such as GRIP (Glutamate Receptor Interacting Protein), ABP (AMPAR-binding protein), SAP97 (synapse-associated protein-97), PICK1 (Protein interacting with C kinase-1), stargazin, NSF (N-ethylmaleimide-sensitive factor), and AP2 (adaptor protein-2)(Xia et al., supra; Dong et al., supra; Osten et al., supra; Daw et al., supra; Chen, L., et al., Nature, 408(6815), 936-43, 2000; Lee, S. H., et al., Neuron, 36(4): 661-74, 2002; Nishimune, A., et al., Neuron, 21(1): 87-97, 1998; Song, I., et al., Neuron, 21(2): 393-400, 1998; Srivastava, S., et al., Neuron, 21(3): 581-91, 1998; Dong, H., et al., J Neurosci, 19(16): 6930-41, 1999). These proteins have been shown to regulate AMPAR function in a variety of ways, including altering AMPAR localization, clustering and/or trafficking.
GAPDH is a tetrameric protein composed of four identical subunits (37 kDa). Each monomer has binding sites for the substrate (glyceraldehyde-3-phosphate, G3P) and the co-factor nicotinamide adenine dinucleotide (NAD+)(Chuang, D. M., et al., Annu Rev Pharmacol Toxicol 45, 269-290, 2005; Sirover, M. A., J Cell Biochem 95, 45-52, 2005). Traditionally, GAPDH has been considered the key enzyme in glycolysis, and therefore, an important protein in energy production. In addition, GADPH was thought to be a housekeeping gene whose transcript level remained constant under most experimental conditions. However, recent evidence supports the notion that GAPDH plays a critical role in apoptosis, as indicated by changes in GAPDH expression and subcellular localization (Sawa, A., et al., Proc Natl Acad Sci USA, 94(21): p. 11669-74, 1997; Ishitani, R., et al., Mol Pharmacol, 53(4): p. 701-7, 1998; Ishitani, R. and D. M. Chuang, Proc Natl Acad Sci USA, 93(18): 9937-41, 1996; Hara, M. R., et al., Nat Cell Biol, 2005. 7(7): 665-74, 2005). GAPDH is overexpressed and accumulates in the nucleus during apoptosis induced by a variety of insults. The mechanism underlying GAPDH nuclear translocation and subsequent cell death remains largely unknown. However, recent studies have implicated several potential factors that may be involved in the process: (1) the expression of GAPDH is regulated by p53, a tumor suppressor protein and proapoptotic transcription factor, which suggests that GAPDH could be a downstream apoptotic mediator (Chen, R. W., et al., J Neurosci 19, 9654-9662, 1999); (2) overexpression of Bcl-2 blocks the apoptotic insults triggered by GAPDH overexpression, nuclear translocation and subsequent apoptosis, suggesting that Bcl-2 may participate in the regulation of GAPDH nuclear translocation, consistent with the anti-apoptic function of Bcl-2 (Dastoor, Z., and Dreyer, J. L., J Cell Sci 114, 1643-1653, 2001); (3) GAPDH binds to a nuclear localization-signal-containing protein, Siah1, which initiates its translocation to the nucleus. The association with GAPDH stabilizes Siah1 and thereby enhances Siah1-mediated proteolytic cleavage of its nuclear substrates and triggers apoptosis (Hara et al., supra; Hara, M. R., and Snyder, S. H., Annu Rev Pharmacol Toxicol., 2006; Hara, M. R., and Snyder, S. H., Cell Mol Neurobiol., 2006; Hara, M. R., et al., Proc Natl Acad Sci USA 103, 3887-3889, 2006).
Based on the foregoing, there is a need in the art for compositions and methods for the treatment and prophylaxis of MS.